Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 10 October 2017 Experimental Completion Date: 10 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
1,1,3,3-Tetramethylbutyl peroxy-2-ethylhexanoate
CAS# 22288-43-3
Specific details on test material used for the study:
Identification: 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3)
Physical State/Appearance: Clear colorless liquid
CAS Number: 22288-43-3
Purity: 99.3%
Batch Number: 1501442030
Label: TRIGONOX 421, 1, 1, 3, 3-Tetramethylbutyl peroxy-2-ethylhexanoate, Storage: -20 degrees C min to +5 degrees C max
Date Received: 27 September 2017
Storage Conditions: Store frozen at approximately -20 °C in the dark; used/formulated at ambient temperature 10 to 30 °C
Expiry Date: 31 July 2018
No correction for purity was made.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of one hundred and twenty animals (sixty males and sixty females) were accepted into the study. At the start of treatment the males weighed 188 to 216g, the females weighed 142 to 173g, and were approximately six weeks old.

Animal Care and Husbandry
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
The animals were randomized and allocated to dose groups on arrival. Subsequent group mean body weights were checked on Day -2 and animals reallocated where necessary to ensure similarity between the dose groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Vehicle:
arachis oil
Details on oral exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-one days. Formulations were therefore prepared either weekly or fortnightly and stored at approximately 4 ºC in the dark.
Samples of test item formulations were taken and analyzed on four occasions for concentration of 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3) at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 7% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test sample was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.

Preparation of calibration standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.1 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent to yield a solution with a concentration of 1 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.1 mg/mL.
On each occasion standard solutions derived from two stock standard solutions were used for claculation.
Calibration standards were injected into the instrument, at the beginning and end of each sample analysis sequence as a minimum.

Preparation of test samples
The formulations received were extracted with extract solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then ultra-sonicated for 30 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of accuracy and precision samples.
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations. These samples were then prepared for analysis.
The concentration of test item in the final solution was quantified by GC using FID detection as detailed in the instrument paraneters below:

HPLC: Agilent Technologies 5890, incorporating autosampler and work station
Column: ZB-5 (30m x 0.53 mm id x 5μ film)
Overn temperature: oven: 50 °C for 1 minute, with 10 °C/minute to 260 °C for 10 minutes.
Injection temperature: 250 °C
Flame ionisation temperature: 1 µL
Retention time: From 4 to 7 mins

The analytical procedure was successfully validated with respects to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity and stability was confirmed for Test item in Arachis Oil BP formulations at nominal concentrations of 2.5 mg/mL and 250 mg/mL when stored refrigerated for 21 days.
The mean concentration of Test item in test formulation analzyed for the study were within ±10 % of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
90 days
Two recovery groups, each of ten males and ten females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females in each group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work (Envigo Research Limited Study Number 41103876; Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the rat (OECD 422)).

Dose administration
Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP. Recovery group animals were maintained for a further twenty-eight days following termination of treatment.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Examinations

Observations and examinations performed and frequency:
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all non-recovery animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each non-recovery animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all animals were examined pre-treatment and for non-recovery control and non-recovery high dose animals before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

Estrous Cycle Assessment
Vaginal smears were taken daily for 21 days, for all non-recovery test and control group females during the final three weeks of dosing. The stage of estrus was recorded for each day.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all non-recovery animals from each test and control group at the end of the study (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 132). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 133. Animals were not fasted prior to sampling.
Urinalytical investigations were performed on all non-recovery test and control group animals during Week 12 and on all recovery group animals during the final week of the recovery period. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.
Hematology
The following parameters were measured on plasma from blood collected into tubes containing potassium EDTA anti-coagulant:
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices:
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count:
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Aspartate aminotransferase (ASAT)
Glucose
Alanine aminotransferase (ALAT)
Total protein (Tot.Prot.)
Alkaline phosphatase (AP)
Albumin
Creatinine (Creat)
Albumin/Globulin (A/G) ratio (by calculation)
Triglycerides (Tri)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)
Gamma-glutamyl transferase (yGT)
Inorganic phosphorus (P)

Urinalysis
The following parameters were measured on collected urine:
Volume
Ketones
Specific Gravity
Bilirubin
pH
Urobilinogen
Protein
Blood
Glucose
Appearance
Sacrifice and pathology:
Necropsy
On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all animals were killed by intravenous overdose of a suitable barbiturate followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded

Sperm Analysis
At necropsy, the left testis and epididymis were removed from all males, dissected from connective tissue and weighed separately.
For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyzer to determine the numbers of motile, progressively motile and non-motile sperm.
For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C.
The cauda epididymis was separated from the body of the epididymis and weighed. The cauda epididymis was frozen at approximately -20°C.
Morphological assessment was performed on a sample of a minimum of 200 sperm, where possible, to determine the number with apparent structural anomalies.
Assessment of morphology was only performed for non-recovery control and non-recovery high dose males. As there were no treatment-related findings, these evaluations were not extended to males from other dose groups.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Ovaries
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus
Liver

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint) (Retained only and not processed)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Cecum
Salivary glands (submaxillary)
Colon
Duodenum
Seminal vesicles
Epididymides (Preserved in Bouin’s fluid)
Skin
Esophagus
Spinal cord (cervical, mid thoracic
Eyes (fixed in Davidson’s fluid)
and lumbar)
Gross lesions
Spleen
Heart
Stomach
Ileum (including Peyer’s patches)
Testes (Preserved in Bouin’s fluid)
Jejunum
Thymus
Kidneys
Thyroid/Parathyroid
Liver
Tongue (Retained only and not processed)
Lungs (with bronchi) (Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative)
Trachea
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus (with cervix)
Muscle (skeletal) (Retained only and not processed)
Vagina
All tissues were dispatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Lewis). All tissues from non-recovery control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. Additional slides for immunohistochemical staining of the kidneys for α-2u-globulin were also performed (from additional sections from both kidneys) for males from the non-recovery control and non-recovery high dose groups at the Test Site for histology processing. The prepared slides were then sent to the Test Site for immunohistochemical staining (Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS, UK, Principal Investigator: R Jenkins) prior to dispatch to the Study Pathologist.
Since there were indications of treatment-related adrenal, kidney, liver, thyroid and spleen changes, examination was subsequently extended to include similarly prepared sections of the adrenals (both sexes), kidneys (males only), liver (both sexes), thyroids (males only) and spleen (both sexes) from animals in the low, intermediate and recovery dose groups.
Sciatic nerve
Statistics:
Please see section "Any other information on materials and methods"

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 1000 mg/kg bw/day showed incidences of increased salivation from Day 7 (females) and Day 8 (males) onwards. Instances of noisy respiration were evident in one male and two females treated with 1000 mg/kg bw/day on one or two occasions. Observations such as increased salivation and noisy respiration are commonly observed following the oral administration of an unpalatable or irritant test item formulation and represent difficulties in dosing particular animals rather than evidence of true systemic toxicity.
Generalized fur loss was evident in one recovery control female between Days 89 and 133. Observations of this nature are commonly observed in group housed animals and are considered to be incidental. Red/brown stained snout was evident in one female treated with 1000 mg/kg bw/day on one occasion. An isolated incident of noisy respiration was evident in one non-recovery control male and one female treated with 10 mg/kg bw/day. One recovery control male had a damaged tail. These observations were considered to be incidental.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a reduction in body weight gain during the first nine weeks of treatment. With the exception of Weeks 2 and 7, statistical significance (p<0.05-0.01) was achieved. Slight improvement was evident during Weeks 10 and 11, however, lower body weight gains were again evident during Week 12 and statistically significantly reduced (p<0.05) during Week 13. During the treatment-free period, slightly lower body weight gains were evident in males previously treated with 1000 mg/kg bw/day during the first and fourth week, however, during the remaining weeks, body weight gains exceeded or were comparable to controls.
No such effects were evident in females treated with 1000 mg/kg bw/day or in animals of either sex treated with 100 or 10 mg/kg bw/day.
Females treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.01) in body weight gain during Week 1 of treatment and females previously treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05) in body weight gain during the second week of the treatment-free period. An increase in body weight gain is considered not to represent an adverse effect of treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in food consumption or food conversion efficiencies.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in water consumption.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes observed during ophthalmoscopic examination of animals of both sexes from the control group and high dose group during Week 12 of the treatment period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 1000 mg/kg bw/day showed statistically significant reductions (p<0.01) in hemoglobin, erythrocyte count, hematocrit and mean corpuscular hemoglobin concentration and a statistically significant (p<0.01) increase in mean corpuscular volume. Males from this treatment group also showed a statistically significant reduction (p<0.05) in activated partial thromboplastin time, whilst females from this treatment group also showed statistically significant increases (p<0.01) in total leucocyte count, neutrophils, lymphocytes and platelet count. Although some individual values were within the historical control ranges, a number of the individual values for these parameters were outside of the historical control ranges for rats of the strain and aged used and the intergroup differences were considered to be related to the microscopic spleen changes evident in either sex at this dosage.
No such effects were evident in animals of either sex treated with 100 or 10 mg/kg bw/day or in recovery animals following the treatment-free period.
Following the treatment-free period, females that were previously given 1000 mg/kg bw/day showed a statistically significant reduction (p<0.05) in eosinophils. All of the individual values were within the historical control range and in the absence of a similar effect in 1000 mg/kg bw/day females at the end of the treatment period the intergroup difference was considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 1000 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in albumin/globulin ratio and aspartate aminotransferase and statistically significant reductions (p<0.05-0.01) in total protein, triglycerides, cholesterol and bilirubin. Females treated with 1000 mg/kg bw/day showed statistically significant increases (p<0.01) in total protein and albumin. With the exception of cholesterol, the individual values for the remaining parameters were within historical control ranges; however, with the associated hepatic changes evident in either sex at this dosage, a relationship to treatment cannot be excluded.
No toxicologically significant effects were evident in animals of either sex treated with 100 or 10 mg/kg bw/day or in recovery animals following the treatment-free period.
Females from all treatment groups showed a statistically significant increase (p<0.01) in sodium concentration. Females treated with 1000 mg/kg bw/day also showed statistically significant increases (p<0.01) in potassium and calcium concentration. All of the individual values for sodium and potassium concentration were within historical control ranges and although the majority of calcium values were above the historical control range, three control values were also above the range. There was no dose-related response for sodium concentration or any associated histopathological correlates, therefore, the intergroup differences were considered not to be of toxicological significance.
Males treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.05) in bilirubin whilst females from this treatment group showed a statistically significant increase (p<0.05) in total protein. All of the individual values were within the historical control ranges and in the absence of any associated histopathological correlates at this dosage, the intergroup differences were considered not to be of toxicological significance.
Females treated with 10 mg/kg bw/day showed a statistically significant reduction (p<0.01) in creatinine. The majority of the individual values were within the historical control range and in the absence of a similar effect at 100 or 1000 mg/kg bw/day or any associated histopathological correlates, the intergroup difference was considered not to be of toxicological significance.
Following the treatment-free period, females that were previously given 1000 mg/kg bw/day showed a statistically significant increase (p<0.01) in bile acids. In the absence of a similar effect in 1000 mg/kg bw/day females at the end of the treatment period the intergroup difference was considered not to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the urinalytical parameters measured.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments
There were no treatment-related changes in behavioral assessments.
Isolated instances of increased salivation were evident in a few females treated with 1000 mg/kg bw/day during Weeks 1, 3 or 4 assessments. This type of observation was also observed during the routine daily clinical observations at this dosage and it was considered to reflect the distaste of the test item formulation rather than true systemic toxicity.

Functional Performance Tests
There were no treatment-related changes in functional performance.
Females treated with 1000 mg/kg bw/day showed a statistically significant reduction (p<0.05) in forelimb grip strength. This was confined to one out of the three tests and in the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be toxicologically significant.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
Description (incidence and severity):
Animals of either sex treated with 1000 mg/kg bw/day showed statistically significant increases (p<0.01) in liver and spleen weights both absolute and relative to terminal body weight. The effect on liver weight continued in females that were previously treated with 1000 mg/kg bw/day. Males treated with 1000 and 100 mg/kg bw/day also showed a statistically significant increase (p<0.01) in absolute and relative kidney weights.
No toxicologically significant effects were detected in females treated with 100 mg/kg bw/day, in animals of either sex treated with 10 mg/kg bw/day or in males previously treated with 1000 mg/kg bw/day.
Animals of either sex treated with 100 mg/kg bw/day and females treated with 10 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in liver weight both absolute and relative to terminal body weight. The majority of individual values were within the historical control ranges and in the absence of any associated histopathological correlates at these dosages, the intergroup differences were considered not to be of toxicological significance.
Females from all non-recovery treatment groups showed a statistically significant increase (p<0.05) in kidney weights both absolute and relative to terminal body weight. The majority of individual values were within the historical control ranges and in the absence of any associated histopathological correlates in this sex, the intergroup differences were considered not to be of toxicological significance.
Males from all non-recovery treatment groups showed statistically significant reductions (p<0.05-0.01) in absolute and relative right epididymis weights and left testis weights. Non-recovery males treated with 1000 mg/kg bw/day also showed statistically significant reductions in left epididymis and right testis weights both absolute and relative to terminal body weights. In the absence of true dose related responses or any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Seven males treated with 1000 mg/kg bw/day had pale kidneys. One of these males also had enlarged kidneys, one also had mottled kidneys and one also had enlarged and mottled kidneys. Two males treated with 100 mg/kg bw/day had mottled kidneys. Seven females treated with 1000 mg/kg bw/day had an enlarged liver. One of these females also had a dark liver. Nine females from this treatment had a dark spleen and seven of these females also had an enlarged spleen.
No toxicologically significant macroscopic abnormalities were detected in females treated with 100 mg/kg bw/day, in animals of either sex treated 10 mg/kg bw/day or in recovery animals following the treatment-free period.
The following macroscopic abnormalities were detected, however, they were either present in the control group or were not associated with any treatment-related findings in the treated groups and therefore were considered to be incidental. Reddened lungs, pale lungs, enlarged adrenals, dark kidneys, a mass (approximately 3 mm x 3 mm) in the vagina, small testes and epididymides, and a dark area on the left lobe of the liver.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related microscopic abnormalities were detected:
Adrenal Glands: Zona glomerulosa hypertrophy was noted at an increased incidence in animals of either sex treated with 1000 mg/kg bw/day. Following the treatment-free period, minimal hypertrophy was present in three males and three females previously treated with 1000 mg/kg bw/day. The incidence and severity of the change had decreased, thus indicating partial recovery.
Kidneys: Hyaline droplets were increased in all males treated with 1000 and 100 mg/kg bw/day. Multifocal basophilic tubules were present in all males treated with 1000 mg/kg bw/day and in nine males treated with 100 mg/kg bw/day along with one male treated with 10 mg/kg bw/day. Proteinaceous casts were present in most males treated with 1000 and 100 mg/kg bw/day. Examination of immunohistochemically-stained sections for α2μ-globulin from control and 1000 mg/kg bw/day males confirmed the increase in hyaline droplets in the kidney tubules of males treated with 1000 mg/kg bw/day compared to controls, although it should be noted that positive staining was present in all sections.
Following the treatment-free period, hyaline droplet accumulation showed complete recovery. Tubular basophilia and proteinaceous casts persisted and occasional instances of interstitial fibrosis and tubular dilation were apparent in all but one male previously treated with 1000 mg/kg bw/day. The severity of the changes had decreased, indicating partial recovery.
Liver: Centrilobular hypertrophy was present in eight males and all females treated with 1000 mg/kg bw/day. Recovery was complete following the treatment-free period.
Spleen: Increased hematopoiesis was present in eight males and all females treated with 1000 mg/kg bw/day, along with three females treated with 100 mg/kg bw/day and in one female treated with 10 mg/kg bw/day. This showed complete recovery following the treatment-free period. Increased hemosiderin was present in one control female and six females treated with 1000 mg/kg bw/day. Following the treatment-free period, it was still present in one control female and eight females previously treated with 1000 mg/kg bw/day.
Thyroid Glands: Follicular cell hypertrophy was present in six males treated with 1000 mg/kg bw/day. This had completely reversed following the treatment-free period.
Other effects:
no effects observed
Description (incidence and severity):
Estrous Cycle Assessment
Assessment of estrous cycles during the final three weeks of treatment for non-recovery females did not indicate any obvious effect of treatment at 10, 100 or 1000 mg/kg bw/day, with all non-recovery females showing regular cycling

Sperm Analysis
No effects on sperm concentration, motility, progressive motility or morphology were observed in treated males when compared to controls.
Statistical analysis of the sperm concentration, motility and progressive motility data did not reveal any significant intergroup differences.

Effect levels

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Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
food consumption and compound intake
water consumption and compound intake
urinalysis
behaviour (functional findings)

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3) for ninety consecutive days, to Wistar rats of both sexes, at dose levels of 10, 100 or 1000 mg/kg bw/day resulted in treatment-related body weight effects in males treated with 1000 mg/kg bw/day, hematological and blood chemical effects in either sex treated with 1000 mg/kg bw/day and microscopic abnormalities in animals of either sex treated with 1000 and 100 mg/kg bw/day. No treatment-related effects were evident in animals of either sex treated with 10 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 10 mg/kg bw/day for both sexes.

The increase hematopoiesis in the spleen of females treated with 100 mg/kg bw/day, in isolation, was considered not to represent an adverse response to treatment; therefore, a No Observed Adverse Effect Level (NOAEL) for females was considered to be 100 mg/gk bw/day.

The kidney effects detected in males treated with 1000 and 100 mg/kg bw/day were considered to represent an adverse effect of the test item, therefore, a 'No Observed Adverse Effect Level' (NOAEL) for males has not been established. However, the kidney changes of hyaline droplets were consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. This effect is, therefore, not indicative of a hazard to human health. In the context of this study, the remaining kidney findings, consisting of basophilic tubules and proteinaceous casts in males are more likely to be correlated to the same condition as hyaline droplets and are, therefore, considered to represent limited relevance to humans. In terms of extrapolation to man and risk assessment calculations whereby effects relating to male rat renal changes are species and sex specific and therefore are not relevant, a NOAEL for males can be established at 100 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

• The OECD Guidelines for Testing of Chemicals No. 408 "Repeated Dose 90-Day Oral Toxicity Study in Rodents” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. Vaginal smears were performed for all non-recovery females during the final three weeks of treatment. Ophthalmoscopic examination was also performed on all animals prior to the start of treatment and on non-recovery control group and non-recovery high dose animals during Week 12.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

There were no unscheduled deaths.

Clinical Observations

Increased salivation was evident in animals of either sex treated with 1000 mg/kg bw/day throughout the treatment period. Isolated incidences of noisy respiration were also evident in a few animals of either sex treated with 1000 mg/kg bw/day. No such effects were evident in animals of either sex treated with 100 or 10 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Males treated with 1000 mg/kg bw/day showed a reduction in body weight gain during the first nine weeks of treatment. Slight improvement was evident during Weeks 10 and 11; however, lower body weight gains were again evident during Weeks 12 and 13. During the treatment-free period, slightly lower body weight gains were evident in males previously treated with 1000 mg/kg bw/day during weeks one and four of the treatment-free period, however, during the remaining weeks, body weight gains exceeded or were comparable to controls. No such effects were evident in females treated with 1000 mg/kg bw/day or in animals of either sex treated with 100 or 10 mg/kg bw/day.

Food Consumption

No effect on food consumption or food conversion efficiency was evident in treated animals when compared to controls.

Water Consumption

Visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmoscopy

Ophthalmoscopic examination of animals of both sexes from the non-recovery control and non-recovery 1000 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Estrous Cycle

Assessment of estrous cycles for non-recovery females during the final three weeks of treatment did not indicate any obvious effect of treatment at 10, 100 or 1000 mg/kg bw/day.

Hematology

Animals of either sex treated with 1000 mg/kg bw/day showed reductions in hemoglobin, erythrocyte count, hematocrit and mean corpuscular hemoglobin concentration and an increase in mean corpuscular volume. Males from this treatment group also showed a statistically significant reduction in activated partial thromboplastin time and females also showed an increase in total leucocyte count, neutrophils, lymphocytes and platelet count. No such effects were evident in animals of either sex treated with 100 or 10 mg/kg bw/day or in recovery animals following the treatment-free period.

Blood Chemistry

Males treated with 1000 mg/kg bw/day showed an increase in albumin/globulin ratio and aspartate aminotransferase and reductions in total protein, triglycerides, cholesterol and bilirubin. Females treated with 1000 mg/kg bw/day showed increases in total protein and albumin. No toxicologically significant effects were evident in animals of either sex treated with 100 or 10 mg/kg bw/day or in recovery animals following the treatment-free period.

Urinalysis

There were no treatment-related effects detected in the urinalytical parameters measured.

Necropsy

Seven males treated with 1000 mg/kg bw/day had pale kidneys. One of these males also had enlarged kidneys, one also had mottled kidneys and one also had enlarged and mottled kidneys. Two males treated with 100 mg/kg bw/day had mottled kidneys. Seven females treated with 1000 mg/kg bw/day had an enlarged liver. One of these females also had a dark liver. Nine females from this treatment group had a dark spleen and seven of these females also had an enlarged spleen. No toxicologically significant macroscopic abnormalities were detected in females treated with 100 mg/kg bw/day, in animals of either sex treated 10 mg/kg bw/day or in recovery animals following the treatment-free period.

Sperm Analysis

No effects on sperm concentration, motility, progressive motility or morphology were observed in treated males when compared to controls.

Organ Weights

Animals of either sex treated with 1000 mg/kg bw/day showed an increase in liver and spleen weights both absolute and relative to terminal body weight. Males treated with 1000 and 100 mg/kg bw/day also showed an increase in absolute and relative kidney weights. No toxicologically significant effects were detected in females treated with 100 mg/kg bw/day, in animals of either sex treated with 10 mg/kg bw/day or in recovery animals following the treatment-free period.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Adrenal Glands: Zona glomerulosa hypertrophy was noted at an increased incidence in animals of either sex treated with 1000 mg/kg bw/day. Following the treatment-free period, minimal hypertrophy was present in three males and three females previously treated with 1000 mg/kg bw/day. The incidence and severity of the change had decreased, thus indicating partial recovery.

Kidneys: Hyaline droplets were increased in all males treated with 1000 and 100 mg/kg bw/day. Multifocal basophilic tubules were present in all males treated with 1000 mg/kg bw/day and in nine males treated with 100 mg/kg bw/day along with one male treated with 10 mg/kg bw/day. Proteinaceous casts were present in most males treated with 1000 and 100 mg/kg bw/day. Examination of immunohistochemically-stained sections for α2μ-globulin from control and 1000 mg/kg bw/day males confirmed the increase in hyaline droplets in the kidney tubules of males treated with 1000 mg/kg bw/day compared to controls, although it should be noted that positive staining was present in all sections.

Following the treatment-free period, hyaline droplet accumulation showed complete recovery. Tubular basophilia and proteinaceous casts persisted and occasional instances of interstitial fibrosis and tubular dilation were apparent in all but one male previously treated with 1000 mg/kg bw/day. The severity of the changes had decreased, indicating partial recovery.

Liver: Centrilobular hypertrophy was present in eight males and all females treated with 1000 mg/kg bw/day. Recovery was complete following the treatment-free period.

Spleen: Increased hematopoiesis was present in eight males and all females treated with 1000 mg/kg bw/day, along with three females treated with 100 mg/kg bw/day and in one female treated with 10 mg/kg bw/day. This showed complete recovery following the treatment-free period. Increased hemosiderin was present in one control female and six females treated with 1000 mg/kg bw/day. Following the treatment-free period, it was still present in one control female and eight females previously treated with 1000 mg/kg bw/day.

Thyroid Glands: Follicular cell hypertrophy was present in six males treated with 1000 mg/kg bw/day. This had completely reversed following the treatment-free period.

Conclusion

The oral (gavage) administration of 1,1,3,3-Tetramethylbutyl 2-Ethylperoxyhexanoate (CAS#22288-43-3) for ninety consecutive days, to Wistar rats of both sexes, at dose levels of 10, 100 or 1000 mg/kg bw/day resulted in treatment-related body weight effects in males treated with 1000 mg/kg bw/day, hematological and blood chemical effects in either sex treated with 1000 mg/kg bw/day and microscopic abnormalities in animals of either sex treated with 1000 and 100 mg/kg bw/day. No treatment-related effects were evident in animals of either sex treated with 10 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 10 mg/kg bw/day for both sexes.

The increased hematopoiesis in the spleen of females treated with 100 mg/kg bw/day, in isolation, was considered not to represent an adverse response to treatment, therefore, a No Observed Adverse Effect Level (NOAEL) for females was considered to be 100 mg/kg bw/day.